E. faecalis, and closely related species, such as E. faecium, have emerged as significant human pathogens, being major etiologic agents of infectious endocarditis, nosocomial infections, burn infections, urinary tract infections, meningitis, and surgical wound infections (Lerwis & Zervos, Eur J. Clin Microbiol Infect Dis 9(2): 111-117, 1990; Moellering Jr., Clin. Infect. Dis. 14(6): 1173-1176, 1992; Megran, Clinical Infect. Dis. 15: 63-71, 1992; Emori & Gaynes, Clin. Microbiol. Rev. 6(4): 428-442, 1993; Jett et al., 1994; Edgeworth et al., Crit. Care Med. 28(8): 1421-1428, 1999; Richards et al., Infection Control Hosp. Epidemiol. 21(8): 510-515, 2000; NNIA System, Am J Infect Control, 32: 470-485, 2004; Biedenbach et al., Diagn. Microbiol. Infect. Dis. 50: 59-69 2004; Linden, Semin. Respir. Crit. Care Med. 28: 632-645, 2007). In terms of oral disease, E. faecalis is the most commonly isolated species from infected root canals of teeth that fail to heal following root canal therapy (Sundqvist et al., Oral Surg. Oral Med. Oral Pathol. Oral Radiol. And Endod. 85(1): 86-93, 1998; Peciuliene et al., J. Endod. 26(10): 593-595, 2000; Pinheiro et al., Int. Endod. J. 36: 1-11, 2003; Siqueira Jr. & Rôças, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 97: 85-94, 2004; Stuart et al., J. Endod. 32(2): 93-98 2006; Zoletti et al., J Endod. 32(8): 722-726 2006).
The existing standard treatment for infections, including those due to enterococci, continues to involve the use of antibiotics. In the case of severe enterococcal infections, the regimen typically includes a cell wall active antibiotic, such as a penicillin or cephalosporin plus an aminoglycoside such as streptomycin (Megran, CID 15:63-71, 1992; Noskin, J Lab Clin Med 130:14-20, 1997). As resistance to these drugs became more common, vancomycin replaced these antibiotics as the drug of choice for treating these infections.
Complicating management of these infections is the development of resistance among many Enterococcal strains against many of the available, previously effective antibiotics, including vancomycin (Harvard et al., Br. Med. J. 1: 688-689, 1959; Murray & Mederski-Samaroj, J. Clin. Invest., 72: 1168-1171, 1983; Uttley et al., Lancet, i: 57-58, 1988; Grayson et al., Antimicrob. Agents Chemother., 35: 2180-2184, 1991; Bonten et al., Lancet Infect. Dis. 1: 314-325, 2001; Tenover & McDonald, Curr. Opin. Infect. Dis. 18: 300-305, 2005). With the appearance of vancomycin resistant enterococci (VREs) that were also resistant to the previously used antibiotics, combinations of vancomycin and quinolone type antibiotics, such as ciprofloxacin, were used, however, quinolone-resistant enterococcal strains also appeared. Although a modest number of new antibiotics, such as linezolid and daptomycin, have been developed to provide treatment alternatives in cases of infection by organisms that are resistant to all previously available antibiotics, there have been numerous reports of resistance by E. faecalis and E. faecium strains to these antibiotics as well (Eliopoulos et al., Antimicrob. Agents Chemother., 45(5): 1088-1092, 1998; Prystowsky et al., Antimicrob. Agents Chemother., 45(7): 2154-2156, 2001; Gonzales et al., Lancet 357(9263): 1179, 2002; Herrero et al., N Eng J Med 346: 867-860, 2002; Johnson et al., Int. J. Antimicrob. Agents 24: 315-319, 2004; Munoz-Price et al., Clin. Infect. Dis., 41: 565-566, 2005; Kanafani et al., Scand. J. Infect. Dis., 39(1): 75-77, 2007; Hidron et al., J Antimicrob. Chemother., 61(6): 1394-1396, 2008; Marshall et al., Microbe, 4(5): 231-238, 2009; Kelesidis et al., Clin. Infect. Dis., 52: 228-234, 2011; Ross et al., J. Chemother., 23(2): 71-76, 2011; Ntokou et al., Antimicrob. Chemother. 67(8): 1819-1823, 2012). Therefore, alternative approaches to manage these infections are desired.
Bacteriophages are bacterial viruses that infect bacterial cells. During their infectious cycle within a host cell, the bacterial virus produces enzymes that will lead to the lysis of the cell and release of progeny virus particles. Harnessing this capacity of the bacteriophage to lyse and kill the host cell may provide a means of controlling antibiotic resistant bacterial infections. This approach, of using bacteriophages to treat and control bacterial infections has several advantages. Bacteriophages are highly specific in that they are only infectious for bacterial cells, and have no capacity for infecting cells of higher life forms such as mammals. In fact, they are so specific that the host range of any one bacteriophage is typically a single bacterial species, or at most, a few closely related bacterial species. Therefore, the effect of any one bacteriophage is limited to a very narrow portion of a mixed bacterial population. This provides an impact on the pathogenic bacteria while leaving the normal bacterial population unaffected. Both antibiotic sensitive and antibiotic resistant bacterial strains can be vulnerable to bacteriophage infection. In addition, in contrast to conventional antibiotics which decrease in concentration in the body after administration, bacteriophage titer can increase after administration, due to proliferation of the virus in the targeted host cell.
The therapeutic potential of bacteriophages was tested in 1919 by d'Herelle, who showed that bacteriophage preparations could be used to successfully treat cases of dysentery (described in Chanishvili, Advances in Virus Res. 83: 3-40, 2012). Further work continued, particularly in eastern Europe, on the use of bacteriophages (“phages”) to treat infectious diseases (Barrow, J Chem Technol Biotechnol 76: 677-682, 2001; Duckworth and Gulig Biodrugs, 16(1): 57-62, 2002, Petty et al. TRENDS in Biotechnol., 25(1): 7-15, 2006; Chanishvili, supra). With the advent of antibiotics in the 1940s, this line of research (phage therapy) fell by the wayside in the west since antibiotics were remarkably effective in combating many bacterial infections. However, in eastern Europe, where availability of antibiotics was limited, research into phage therapy continued, particularly in the Soviet Union, Georgia, and Poland. Here, the therapeutic use of phages became an accepted modality for treating a wide variety of bacterial infections.
Since the 1980s, as antibiotic resistance in pathogenic bacteria began to develop, and become more common in the West, there has been a resurgence in interest in using phages to treat human and animal infections (Summers, Annu. Rev. Microbiol. 55: 437-451, 2001, Alisky et al., J. of Infec. 36: 5-15, 1998, Pirisi, Lancet. 356: 1418, 2000; Ho, Perspectives in Biology and Medicine, 44(1): 1-16, 2001; Merril et al., Naure Revs. Drug Disc. 2: 489-497, 2003; Bradbury, Lancet. 363: 624-625, 2004; Dixon, Lancet Infect Dis. 4: 186, 2004; Schoolnik et al., Nature 22(5): 505-506, 2004; Thiel, Nature 22(1): 31-36, 2004; Skurnik and Strauch, Int. J. Med. Microbiol. 296: 5-14, 2005).
Several recent studies report successful implementation of phage therapy (using either infectious bacteriophages or phage products) in modifying bacterial infections in animals by Acinetobacter baumanii, Escherichia coli, group A streptococci, Enterococcus faecium, Bacillus anthracis, and Pseudomonas aeruginosa (Soothill, J. Med. Microbiol. 37: 258-261, 1992; Merril et al., Proc. Natl. Acad. Sci. USA. 93: 3188-3192, 1996; Nelson et al., Proc. Nat. Acad. Sci. 98(7): 4107-4112, 2001; Biswas et al., Infect. Immun. 70(1): 204-210, 2002; Schuch et al., Nature 418: 884-889, 2002; Watanabe et al., Antimicrob. Agents Chemother. 51: 446-452, 2007). In this regard, it is significant to note that in a study reported by Smith and Huggins, J Gen Microbiol 128: 307-318 (1982), a single intramuscular (IM) dose of phage was more effective in protecting mice from normally lethal IM or intracerebral injections of Escherichia coli or Salmonella enterica, than multiple IM injections of antibiotics such as tetracycline, ampicillin, chloramphenicol, or trimethoprim plus sulfisoxazole. In addition, in the first controlled trial of phage therapy in humans, it was shown that a cocktail of six Pseudomonas aeruginosa bacteriophages effectively treated antibiotic-resistant chronic otitis (Wright et al, Clin. Otolaryngol. 34: 349-357, 2009).
In terms of phage therapy to treat E. faecalis infections, there has been relatively little reported. In 2004, Paisano et al., Oral Microbiol Immunol, 19: 327-330 reported that they could reduce the level of infection of a single E. faecalis strain in an infected dental root canal (in vitro), to an undetectable level, using a bacteriophage preparation. However, the bacteriophage used in this study was not characterized in any way (no morphological description, no genomic analysis).
Isolation of a bacterial virus (phage φEF24C) that could protect mice from otherwise lethal doses of E. faecalis has been reported (Uchiyama et al., FEMS Microbiol Lett. 278: 200-206, 2008; Uchiyama et al., Appl Environ Microbiol. 74(13): 4149-4163, 2008). This bacteriophage was reported to have a broad range of activity against many strains of E. faecalis, and have no untoward effects on the mice. This phage was well characterized and could be described as follows: φEF24C has a contractile tail, giving it the morphology of a Myovirdae type bacteriophage. Its genome consisted of a linear, double stranded DNA, 142,072 by in length, with an estimated 221 ORFs and 5 tRNA genes.
Other strategies for exploiting bacteriophages for controlling E. faecalis infections involve the use of lytic enzymes produced by the viruses to lyse and kill the bacterial cells. The cell lysis produced by these enzymes is needed by the virus in order to allow the release of progeny viral particles from the infected cells. The strategy for exploiting these bacteriophage-specified lytic enzymes involves the cloning and expression of the genes for these enzymes, followed by the purification of the expressed proteins. One such lytic enzyme, active against strains of E. faecalis (as well as strains of E. faecium, and several Streptococcus species), has reportedly been isolated from E. faecalis bacteriophage φ1 (Yoong et al., J Bacteria 186(145): 4808-4812, 2004). The bacteriophage source of this enzyme, phage φ1, was described as a Myoviridae morphotype; that is, a bacteriophage with a contractile tail. A second report of a bacteriophage lytic enzyme active against strains of E. faecalis came from Son et al., Appl. Microbiol. 108: 1769-1779 (2010). Here, the gene for a putative lytic enzyme specified by E. faecalis bacteriophage EFAP-1 was cloned, and expressed, and the gene product was purified. The purified phage protein was found to have lytic activity against numerous strains of E. faecalis and E. faecium. Bacteriophage EFAP-1, the source of the lytic enzyme described by Son et al., had the non-contractile tail structure of a Siphovirdae morphotype. EFAP-1 had a 21,115 bp genome containing 24 ORFs.
Several other bacterial viruses that infect strains of E. faecalis have been reported. These include: Bacteriophages φFC1 (Yang et al., J. Bacteriol. 184: 1859-1864, 2002), F4 (Nigutova et al., Folio Microbiol. 53(3): 234-236, 2008), phages 31, 42, 54, and 70 (Mazaheri Nezhad Fard et al., Curr Microbiol. 60: 400-406, 2010), VD13 (Ackermann et al., Can. J. Microbiol., 21: 571-574, 1975), phages 1 and 2 (Rogers and Sarles, J. Bacteriol. 85: 1378-1385, 1963), SAP6 (Lee and Park, J. Virol. 86(17): 9538-9539, 2012), BC-611 (Horiuchi et al., J. Virol. 86(17): 9538-9539, 2012), and phages φFL1A, φFL1B, φFL1C, φFL2A, φFL2B, φFL3A, φFL3B and φFL4A (Yasmin et al., J. Bacteriol. 192(4): 1122-1130, 2010). In addition several unnamed E. faecalis bacteriophages have been reported (Natkin, Arch Oral Biol. 12(5): 669-680, 1967, Timperley et al., J. Pathol. Bacteriol. 9: 631-634, 1966, Follett et al., J. Gen. Virol. 1: 281-284, 1967, Letkiewicz et al., Folio Microbiol. 54: 457-461, 2009, and Bachrach et al., Lett. Appl. Microbiol. 36: 50-53, 2003). However, none of these have been proposed for use in phage therapy.
φEf11 is a temperate bacteriophage that was induced from a lysogenic root canal isolate of Enterococcus faecalis (Stevens et al., Oral Microbiol. Immunobiol., 24: 278-284, 2009). φEf11 prophage is widely disseminated among strains of E. faecalis. It is a member of the Siphoviridae family, with a long (130 nm) non-contractile tail and a small (41 nm diameter) spherical/icosahedral head. The phage produces small, turbid plaques in lawns of E. faecalis JH2-2. The φEf11 DNA has been sequenced and annotated, disclosing a genome of 42,822 base pairs encoding 65 Open Reading Frames (Stevens et al., FEMS Microbiol. Lett., 317: 9-26, 2011, incorporated herein by reference; GenbankGQ452243.1, incorporated herein by reference).
The φEf11 genome is shown in FIG. 10. The numbered arrows indicate ORFs. The ORF numbering scheme in FIG. 10 corresponds to the numbering system contained in Stevens et al., 2011, supra. ORFs 25-29 are involved in host cell lysis.
φEf11 possesses several characteristics making it a favorable candidate virus to be used in phage therapy: There are no toxin-related genes detected in the φEf11 genome, and it encodes several (4-6) genes encoding proteins with lysis-associated functions (Stevens et al., 2011, supra). However, as a temperate virus that has a very limited host range, and is difficult to propagate, wild-type φEf11 would not be suitable as a potential therapeutic agent.
Moreover, since φEf11 is a temperate bacteriophage, it possesses a module of genes that allows it to integrate its DNA into the host cell chromosome rather than initiating a productive infection and lysing the infected cell. The bacteriophage DNA can remain in this integrated state indefinitely, and the infected cell (a lysogen) will survive and continue to multiply. Furthermore, regulatory elements in the φEf11 genome whose activation is required for the development of a productive/lytic infection within the cell, are inactivated by a protein (repressor) produced by one of the lysogeny-related genes. Therefore, lysogenic cells producing this repressor are immune to super infection by φEf11, and would consequently survive exposure to this virus. This further limits the utility of φEf11 as therapeutic agent